Sialic acid is the only sugar that contains a net negative charge and is typically found on terminating branches of N-glycans, O-glycans, and glycosphingolipids (gangliosides) (and occasionally capping side chains of GPI anchors). The sialic acid modification of cell surface molecules is crucial for many biological phenomena including protein structure and stability, regulation of cell adhesion, and signal transduction. Sialic acid deficiency disorders such as Hereditary Inclusion Body Myopathy (HIBM or HIBM type 2), Nonaka myopathy, and Distal Myopathy with Rimmed Vacuoles (DMRV) are clinical diseases resulting from a reduction in sialic acid production.
HIBM is a rare autosomal recessive neuromuscular disorder caused by a specific biosynthetic defect in the sialic acid synthesis pathway. Eisenberg et al., Nat. Genet. 29:83-87 (2001). The disease usually manifests between the ages of 20 to 40 with foot drop and slowly progressive muscle weakness and atrophy. Patients may suffer difficulties walking with foot drop, gripping and using their hands, and normal body functions like swallowing. Histologically, it is associated with muscle fiber degeneration and formation of vacuoles containing 15-18 nm tubulofilaments that immunoreact like β-amyloid, ubiquitin, prion protein and other amyloid-related proteins. Askanas et al., Curr Opin Rheumatol. 10:530-542 (1998). Both the progressive weakness and histological changes initially spare the quadriceps and certain other muscles of the face. However, the disease is relentlessly progressive with patients becoming incapacitated and wheelchair-confined within two to three decades. There are no treatments currently available.
Studies of an Iranian-Jewish genetic isolate mapped the mutation associated with HIBM to chromosome 9p12-13. Argov et al., Neurology 60:1519-1523 (2003). The causative mutations were identified for HIBM in the gene GNE, which encodes the bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK). Eisenberg et al., Nat. Genet. 29:83-87 (2001). DMRV is a Japanese variant, allelic to HIBM. Nishino et al., Neurology 59:1689-1693 (2002).
The biosynthesis steps and feedback regulation of GNE/MNK is depicted in FIG. 1. The production of sialic acid on glycoconjugates requires the conversion of N-acetylglucosamine (conjugated to its carrier nucleotide sugar UDP) to sialic acid. The sialic acid subsequently enters the nucleus where it is conjugated with its nucleotide sugar carrier CMP to make CMP-sialic acid, which is used as a donor sugar for glycosylation reactions in the cell. CMP-sialic acid is a known regulator of GNE/MNK activity. Jay et al., Gene Reg. & Sys. Biol. 3:181-190 (2009). Patients with HIBM have a deficiency in the production of sialic acid via the rate controlling enzyme GNE/MNK, which conducts the first two steps of this sequence: 1) epimerization of the glucosamine moiety to mannosamine with release of UDP, and 2) phosphorylation of the N-acetylmannosamine. The mutations causing HIBM occur in the regions encoding either the epimerase domain (GNE) or the kinase domain (MNK). Nearly twenty GNE mutations have been reported in HIBM patients from different ethnic backgrounds with founder effects among the Iranian Jews and Japanese. Broccolini et al., Hum. Mutat. 23:632 (2004). Most are missense mutations and result in decreased enzyme GNE activity and underproduction of sialic acid. Sparks et al., Glycobiology 15(11):1102-10 (2005); Penner et al., Biochemistry 45:2968-2977 (2006).
Knock-out of the GNE/MNK gene in mice is lethal as no sialic acid is incompatible with life, but knock-in introduction of human mutant forms of GNE/MNK have allowed the production of mouse models with human disease features. In the DMRV-HIBM mouse model in which Gne-deficient mice transgenically express the human GNE gene with D176V mutation (Gne−/− hGNED176V-Tg), these mice show hyposialylation in various organs in addition to the characteristic features of muscle atrophy, weakness and degeneration, and amyloid deposition. In these mice, hyposialylation is documented from birth, yet the mice only develop muscle symptoms several weeks later, including decreased twitch force production in isolated muscles starting at 10 weeks of age and impairment of motor performance from 20 weeks of age onward. Muscle atrophy and weakness were, however, reduced or prevented after treatment with administration of a sialic acid precursor N-acetylmannosamine (ManNAc), sialic acid, or sialyl-lactose, in water. Malicdan et al., Nat. Medicine 15(6):690-695 (2009). All three sialic acid metabolite tested showed similar treatment effects. In another mouse model of HIBM in which knockin mice harbor the M712T Gne/Mnk mutation, mice homozygous for the M712T Gne mutation died within 72 hours after birth, but lacked a muscle phenotype. Galeano et al., J. Clin. Investigation 117(6) 1585-1594 (2007). Homozygous mice, however, did have severe glomerular hematuria and podocytopathy, including effacement of the podocyte foot processes and segmental splitting of the glomerular basement membrane (GBM). Administration of ManNAc in water to mutant mice improved survival, improved renal histology including less flattened and fused podocyte foot processes, increased sialylation of renal podocalyxin, and increased sialylation of brain PSA-NCAM. Galeano et al., J. Clin. Investigation 117(6) 1585-1594 (2007).
Theoretically, the replacement of any metabolite after the genetic block in the pathway could alleviate symptoms of a sialic acid deficiency if the production of sialic acid is the key reason the mutation causes the disease. Jay et al., Gene Reg. and Sys. Biology 3:181-190 (2009). The challenge in administering a compound in the sialic acid biosynthetic pathway in vivo, however, is mainly its rapid clearance and excretion in the urine. After a single intraperitoneal injection of N-acetylneuraminic acid (NeuAc), the sialic acid concentration in the serum was considerably increased within minutes, but 90% of the sialic acid was found in the urine within 5-30 min, and almost all of it was excreted within 4 hours. After a single dose of NeuAc by the intragastric route, the sialic acid concentration in the serum was half that achieved by the intraperitoneal route, but the excretion rate was slower, as 70% of the sialic acid was found in the urine within 30-60 min. A similar pattern of rapid excretion was observed after a single dose of the physiological sialic acid precursor, ManNAc. Malicdan et al., Nat. Medicine 15(6):690-695 (2009).
Treatment experiments by Galeano et al. and Malicdan et al. described above utilized exposure to the drug via water intake which provides a longer term exposure to drug than might be achieved by bolus or episodic treatment. Continuous treatment, such as continuous water-based exposure, is not reasonable or preferred in human treatment due in part to the logistics of performing it and the resulting difficulty with medication compliance.
Further, the treatment of sialic acid deficiencies is complicated by the fact that each individual may carry a different GNE mutation, which may affect either the epimerase domain (GNE) and/or kinase domain (MNK) of the enzyme and to varying degrees. The residual catalytic activity of the MNK might perform this function, but not in all patients. If ManNAc is administered to an individual who lacks most or all MNK activity, treatment will be dependent on unknown kinases or N-acetylglucosamine kinase to transform ManNAc to ManNAc-6-phosphate. The degree and quantity of these other kinase enzymes and their efficiency in transforming ManNAc to the phosphorylated form is unknown and will likely vary between people. This process may be slow or unpredictable and could delay or limit the onset of sialic acid production in some patients relative to giving sialic acid directly. ManNAc may have an advantage if its absorption into cells is improved due to its lack of charge. Its distribution to some tissues or in some people may be better than charged sialic acid if the variable kinase and metabolic steps as well as the rapid clearance are not limiting. ManNAc is a direct product of GNE/MNK and so may also act as a typical product inhibitor of the residual enzyme activity causing a decrease in endogenous production. This phenomenon may explain the very flat therapeutic effect curve showing that 20 mg/kg, 200 mg/kg and 2,000 mg/kg had very similar levels of efficacy which was still not completely effective in preventing disease in mouse DMRV model experiments. Malicdan et al., Nat. Medicine 15(6):690-695 (2009).
Sialic acid as treatment avoids the need for the random kinase phosphorylation of ManNAc and is more immediately available. Since it avoids the uncertainty of the kinase step, sialic acid may provide a better replacement efficacy if the genetics and phenotype of the patient make phosphorylation less efficient in a given individual and to minimize tissue-dependent phosphorylation variations. Sialic acid's action will likely be more immediate as it is near the end of the sialic acid biosynthetic pathway; however, sialic acid is also quickly cleared primarily by the kidneys. Therefore, sialic acid may have rapid-on and rapid-off effects. In addition, giving too much of sialic acid could result in a surge in CMP-sialic acid which could act as a potential feedback inhibitor of the GNE/MNK enzyme, which could have negative consequences on overall biosynthesis of sialic acid production and worsen symptoms of the sialic acid deficiency. Jay et al., Gene Reg. and Sys. Biology 3:181-190 (2009).
Effective replacement of substrate within the sialic biosynthetic pathway may require a more steady even exposure to non-inhibiting or regulating levels of sialic acid metabolites, while at the same time maintain an adequate pool of CMP-sialic acid for glycosylation reactions. Several data suggest that the production of CMP-sialic acid is highly regulated and that there is not a large pool of CMP sialic acid allowed to accumulate. The homology of mouse and human GNE/MNK is about 98% which is far above the normal levels of homology, suggesting very tight control of this enzyme's function. Seppala et al., Am. J. Hum. Genet. 64: 1563-1569 (1999). The allosteric regulation by CMP-sialic acid further suggests that the system is finely tuned to assure steady production of sialic acid and not excess production. Mutations in GNE/MNK that interfere with this regulation cause a disease called sialuria due to excess sialic acid production. Since sialic acid is prepared for attachment to biologicals as a high energy nucleotide carrier CMP, it is certainly expected that excess use of a high energy intermediate would not be optimal for the cell. Finally, data from pharmacokinetics and metabolism experiments suggest that very small percentages of administered sialic acid or ManNAc are incorporated into the body after a dose, and the vast majority is excreted and not stored. Given the lack of a significant pool, dietary sialic acid or mannosamine is insufficient to maintain glycosylation through the day and night. The muscle is most limited since its expression of GNE/MNK is very low compared to other tissues like the liver. Given the muscle phenotype of mutations, it is reasonable to assume that biosynthesis is tightly regulated to sialylation needs, and that no substantial pool exists in vivo. In this situation, effective delivery of CMP-sialic acid to the sites of glycosylation in the cell requires a steady and continuous exposure to assure that feedback regulation is not induced by spikes in concentration, and that glycosylation does not become deficient during periods of low substrate. This low period may be particularly problematic at night, when intake of sialic acid or metabolites is not occurring, and ⅔rds of the growth hormone and insulin-like growth factor 1 (IGF-1) is produced, which is critical for inducing muscle repair and anabolism. Frost and Lang Minerva, Endocrinol. 28:53-73 (2003); Wajnrajch J., Pediatr. Endocrinol. Metab. 18:325-338 (2005). Effective substrate replacement may require then continuous and steady provision of the metabolites to many tissues and be effective in a wide variety of patient types with different mutations.
Given the need for continuous exposure and the plasma half-lives of sialic acid biosynthetic pathway components, there is a need for formulations which provide extended exposure to sialic acid, reduce immediate surges in the metabolites of sialic acid, have general tissue availability, and are efficacious in individuals across a broad range of genotypes/phenotypes.
All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.